Monkey-origin embryonic stem cells

ABSTRACT

A method for producing a monkey-derived embryonic stem cell comprising the steps of carrying out fertilization by insemination by in vitro fertilization or intracytoplasmic sperm injection using a monkey ovum and monkey sperms, thereby giving a fertilized ovum, allowing the fertilized ovum to differentiate into a blastocyst by in vitro culture, and establishing an ES cell line using the blastocyst; the monkey ES cell obtained by the method, a method for screening a reagent for specific differentiation into cell or tissue by using the ES cell; and a differentiated cell or differentiated tissue each differentiated from the ES cell. According to the present invention, applications of the embryonic stem cells to embryological studies clinical applications, experimental models, and the like on primates, studies of diseases, are expected.

TECHNICAL FIELD

[0001] The present invention relates to a monkey-derived embryonic stemcell which is useful for embryological studies, studies of diseases,clinical applications, experimental models, and the like on primates,particularly on human and a monkey; a method for producing amonkey-derived embryonic stem cell, by which the monkey-derivedembryonic stem cell can be obtained in a high yield; a method forscreening a reagent for specific differentiation into a tissue or acell, which is useful for obtaining the desired differentiated cell orthe desired differentiated tissue; and a differentiated cell ordifferentiated tissue.

BACKGROUND ART

[0002] An embryonic stem cell (hereinafter also referred to as ES cell)is an undifferentiated cell having the pluripotency and the ability ofself-replication. In addition, it is suggested that the ES cell has theability to repair damaged tissues. Therefore, intensive studies on EScells have been conducted, since the ES cells are useful in thescreening of therapeutic substances for various diseases, the field ofregenerative medicine, and the like.

[0003] Currently, mouse-derived ES cells have been widely used in, forinstance, the production of an engineered mouse in which a specific genehas been altered by gene targeting, and the like. However, in the caseof the application of mouse-derived ES cells on the models of humandiseases, the expected effects may not always be obtained for thefollowing reasons: a) there are some genes expressed at different stagesbetween mouse embryos and human embryos; b) the structures or functionsof the extraembryonic tissues such as the placentas are different; c)the structures of the embryonic tissues of the early-embryos nidated aredifferent.

[0004] On the other hand, monkey-derived embryonic stem cells aresuitable for use for the studies of human diseases, because they aremore closely related to human, as compared to mouse-derived ES cells.

[0005] Although approximately 200 kinds of monkeys have beenconventionally known around the world, the types of monkeys used for theroutine experiments are limited in the current situations. Higherprimates can be roughly divided into the following two groups:

[0006] (1) New World Primates

[0007] The common marmoset (Callithrix jacchus) has been widely known,and used as an experimental primate. The development of the New Worldprimate is basically similar to that of the Old World primate, with somedifferences regarding the structures of the embryos or the placentas.

[0008] (2) Old World Primates

[0009] The Old World primate is a primate extremely closely related tohuman. The rhesus monkey (Macaca mulatta) and the cynomolgus monkey(Macaca fascicularis) are known. The Japanese monkey (Macaca fuscata)belongs to the same genus (the genus Macaca) as Macaca fascicularis. Thedevelopment of the Old World primate is extremely similar to that ofhuman.

[0010] Currently, as monkey-derived ES cells, the common marmoset EScells [Thomson, J. A. et al., Biol. Reprod. 55, 254-259 (1996)], and therhesus ES cells [Thomson, J. A. et al., Proc. Natl. Acad. Sci. U.S.A.92, 7844-7848 (1995)] have been established. However, as describedabove, Callithrix jacchus belongs to the New World primates which arenot genealogically derived from humans, with some differences in thestructures of the embryos or the placentas. Further, since Callithrixjacchus is small, the manipulations in various experiments are not easyin the current situations, and there are little background data. On theother hand, Macaca mulatto is rarely used as an experimental animal inJapan and Europe. In addition, the reproduction is seasonal, so that theregular ovulation does not necessarily occur throughout the year.Furthermore, there are defects in preparation of the common marmoset EScells and the rhesus ES cells that the collection of ova istime-consuming and inefficient.

[0011] Although human ES cells also have been developed, the use thereofmay be limited from the ethical viewpoint.

DISCLOSURE OF INVENTION

[0012] The present invention has been accomplished in light of the priorart described above. An object of the present invention is to provide amonkey-derived embryonic stem cell which is useful for embryologicalstudies, studies of diseases, clinical applications, experimentalmodels, and the like on primates, particularly on human and a monkey; amethod for producing the monkey-derived embryonic stem cell, by whichthe monkey-derived embryonic stem cell can be obtained in a high yield;a method for screening a reagent for specific differentiation into acell or tissue, which is useful for obtaining the desired differentiatedcell or the desired differentiated tissue; and a differentiated cell ordifferentiated tissue.

[0013] The gist of the present invention relates to

[0014] [1] a monkey-derived embryonic stem cell obtainable by carryingout a process comprising:

[0015] (a) carrying out fertilization by in vitro fertilization orintracytoplasmic sperm injection using a monkey ovum and a monkey sperm,thereby giving a fertilized ovum;

[0016] (b) allowing the fertilized ovum obtained in step (a) todifferentiate into a blastocyst by in vitro culture; and

[0017] (c) establishing an embryonic stem cell line using the blastocystobtained in step (b);

[0018] [2] a method for producing a monkey-derived embryonic stem cell,comprising:

[0019] (a) carrying out fertilization by in vitro fertilization orintracytoplasmic sperm injection using a monkey ovum and a monkey sperm,thereby giving a fertilized ovum;

[0020] (b) allowing the fertilized ovum obtained in step (a) todifferentiate into a blastocyst by in vitro culture; and

[0021] (c) establishing an embryonic stem cell line using the blastocystobtained in step (b);

[0022] [3] an established cynomolgus monkey-derived cell having thefollowing properties:

[0023] (i) being capable of indefinite proliferation while maintainingan undifferentiated state;

[0024] (ii) having the same chromosome number as that of original Macacafascicularis;

[0025] (iii) exhibiting the pluripotency when transplanted tohypodermis, the renal capsule or the testis of a 8-12 week-old SCIDmouse or nude mouse;

[0026] (iv) being negative for SSEA-1 and positive for SSEA-3 andSSEA-4; and

[0027] (v) exhibiting an alkaline phosphatase activity;

[0028] [4] a method for screening a reagent for specific differentiationinto a tissue or a cell, characterized by maintaining a cell selectedfrom the group consisting of the monkey-derived embryonic stem cellaccording to item [1] above and the cynomolgus monkey-derived cellsaccording to item [3] above, in the presence of a test substance; and

[0029] [5] a differentiated cell or differentiated tissue, resultingfrom differentiation of an embryonic stem cell selected from the groupconsisting of the embryonic stem cell according to item [1] above andthe cynomolgus monkey-derived cell according to item [3] above.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030]FIG. 1 is a photograph showing the results of microscopicobservation of the cynomolgus monkey-derived embryonic stem cells of thepresent invention. The upper panel shows the results of observation at alow magnification (100×), and the lower panel shows the results ofobservation at a high magnification (200×).

[0031]FIG. 2 is a photograph showing the results of observation of thecynomolgus monkey-derived embryonic stem cells of the present inventionby various techniques. Panel A and Panel B respectively show the resultsof 0microscopic observations of the cynomolgus monkey-derived embryonicstem cells of the present invention [Panel A: low magnification (Bar=100μm), Panel B: high magnification (Bar=50 μm)]. Panel C shows the resultsof microscopic observation of the cynomolgus monkey-derived embryonicstem cells of the present invention after alkaline phosphatase staining(Bar=100 μm). Panel D shows the results of microscopic observation ofthe cynomolgus monkey-derived embryonic stem cells of the presentinvention after immunostaining for SSEA-4 (Bar=100 μm).

[0032]FIG. 3 is a photograph showing the results of microscopicobservations of the tumors after various stainings, wherein the tumorswere formed by injecting subcutaneously the cynomolgus monkey-derivedembryonic stem cells of the present invention into a mouse. Panels A toH show the results of microscopic observations of the tumors after HEstaining, wherein the tumors were formed by injecting subcutaneously thecynomolgus monkey-derived embryonic stem cells of the present inventioninto a mouse. Panel A: whole tumor (Bar=300 μm), Panel B:neuroepithelium (Bar=200 μm), Panel C: glia (Bar=200 μm), Panel D: gland(Bar=200 μm), Panel E: muscle (Bar=200 μm), Panel F: cartilage (Bar=400μm), Panel G: bone (Bar=200 μm), and Panel H: ciliated epithelium(Bar=150 μm). Panels I to M show the results of microscopic observationsof the above-mentioned tumors after immunostaining. Panel I:immunostaining of neuron and glia for NSE (Bar=200 μm), Panel J:immunostaining of glia for GFAP (Bar=200 μm), Panel K: immunostaining ofperipheral nerve for NSE (Bar=200 μm), Panel L: immunostaining of musclefor desmin (Bar=200 μm), and Panel M: immunostaining of cartilage forS-100 proteins (Bar=400 μm).

BEST MODE FOR CARRYING OUT THE INVENTION

[0033] The “monkey” used in the present invention refers to primates,specifically the New World primates and the Old World primates. Amongthem, the Old World primates are expected to be used as model animals ofdiseases close to human, or used in a system for screening therapeuticagents for various diseases, because they are primates which areextremely closely related to human, and developmentally resemble human.Therefore, in the present invention, the Old World primates,specifically including Macaca fuscata and Macaca fascicularis, aredesirable, and Macaca fascicularis is especially preferable.

[0034] The above-mentioned Macaca fuscata and Macaca fascicularis havecloser genealogical relations to humans. Since Macaca fuscata ismiddle-sized (weight: 5 to 15 kg), there are advantages in that asurgical operation can be easily performed on Macaca fuscata, and thatMacaca fuscata has sufficient physical strength. Further, Macaca fuscatahas docile nature and training of Macaca fuscata is greatly successful.Therefore, it is advantageous in that various experiments can be carriedout under no anesthesia. On the other hand, since Macaca fascicularis issmall (weight: 3 to 6 kg), the Macaca fascicularis has advantages suchthat the handling thereof in various animal experiments is easy, andthat there are many examples of the use of the Macaca fascicularis as alaboratory animal in Japan and Europe, and many background data areobtained. In addition, ovulation occurs throughout the year in Macacafascicularis, so that it is more advantageous in genealogical andphysiological experiments than Macaca mulatta with seasonal ovulation.

[0035] The monkey-derived embryonic stem cell of the present inventioncan be prepared by a process (hereinafter referred to as a method forproducing a monkey-derived embryonic stem cell) comprising:

[0036] (a) carrying out fertilization by in vitro fertilization orintracytoplasmic sperm injection using a monkey ovum and a monkey sperm,thereby giving a fertilized ovum;

[0037] (b) allowing the fertilized ovum obtained in the step (a) todifferentiate into a blastocyst by in vitro culture; and

[0038] (c) establishing an embryonic stem cell line using the blastocystobtained in the step (b).

[0039] The method for producing a monkey-derived embryonic stem cell isencompassed by the present invention.

[0040] The method for producing a monkey-derived embryonic stem cell ofthe present invention is based on the findings of the present inventorsthat a blastocyst can be obtained from a fertilized ovum in asurprisingly high probability of about 40 to 46%, since the steps (a) to(c) described above are carried out. Thus, according to the method ofthe present invention, there can be also exhibited an excellent effectthat a monkey-derived blastocyst can be developed in an excellentefficiency. Therefore, according to the method of the present invention,there is exhibited an excellent effect that a monkey-derived embryonicstem cell can be obtained in an extremely high yield, as compared toconventional methods (e.g., WO 96/22362 and the like).

[0041] In the step (a), monkey ova can be collected by a method ofperforming abdominal dissection and ovariocentesis under direct vision,a method of extracting a uterine tube and then washing it, to collectovulated ova, or other methods. It is desired that ova are collectedfrom a monkey under laparoscopic observation, from the viewpoints ofreducing the burden on the animal, shortening or eliminating the timerequired for wound healing after the surgical treatment and reducing therisk for infection in the animal. Ovum collection under laparoscopicobservation may be carried out, for instance, by making an incision ofonly about 1 cm, inserting a laparoscope, and performing ovariocentesisthrough the incision in the abdominal wall. Since magnified views of thelocal site are easily obtained by this process, it is advantageous inthat the centesis site can be targeted more accurately to collect ova,as compared to the case where ovariocentesis is performed under directvision. In addition, collection of an ovum under laparoscopicobservation is desirable from the viewpoint of animal welfare, becauseonly one stitch is required to suture the abdominal wall after the ovumcollection and the operation can be completed in a short time.

[0042] The age of the female monkey used for ovum collection may varydepending on the kind of the monkey. It is desired that the age of thefemale monkey is 3.5 years old or more, preferably 4 years old or more,from the viewpoint that it is desired to show regular menstrual cycle.In addition, it is desired that the age of the females are 20 years oldor less, preferably 15 years old or less, from the viewpoint that theyare before the menopause. Specifically, it is desired that Macacafuscata is 5 to 15 year-old and Macaca fascicularis is 4 to 15 year-old.

[0043] For ovum collection, an ovulation-inducing agent may be used. Theovulation-inducing agent includes follicle-stimulating hormone (FSH),luteinizing hormone (LH), gonadotropin releasing hormone (GnRH), and thelike, and specifically, for example, gonadotropin releasing hormone(GnRH), pregnant mare's serum gonadotropin (PMSG), human menopausalgonadotropin (hMG), human chorionic gonadotropin (hCG), luteinizinghormone-releasing hormone (LHRH), follicle-stimulating hormone (FSH),and the like. The amount and time period of the ovulation-inducing agentadministered can be appropriately selected depending on the weight ofthe individual, the kind of the ovulation-inducing agent used, withinthe range in which the ovulation-inducing effects are exhibited.

[0044] It is desired that the monkey ovum used in the step (a) describedabove is mature and at the MII stage, and that the cytoplasm of the ovumis homogeneous and elastic. Such properties can be evaluated byobserving the process of the intracytoplasmic sperm injection or invitro fertilization.

[0045] The ovum collection under laparoscopic observation can be carriedout as specifically described below: The amount 1.8 to 3.65 mg ofgonadotropin releasing hormone (GnRH) is administered subcutaneously to5 to 15 year-old females of Macaca fuscata or 4 to 15 year-old femalesof Macaca fascicularis. Two weeks after the administration of GnRH, 25IU/kg of pregnant mare's serum gonadotropin (PMSG), 10 IU/kg of humanmenopausal gonadotropin (hMG) or 3 IU/kg of follicle-stimulating hormone(FSH) is administered intramuscularly once a day at a given time forconsecutive 9 days. Four to five days after the administration, theovaries are observed using a laparoscope (outer diameter: 3 mm), therebyconfirming whether the ovarian follicles develop or not. Here, whetherthe ovarian follicles develop or not is evaluated using as an index thefact that a plurality of bodies with slightly swollen albuginea areformed in the ovary, whereby the ovary as a whole is enlarged and theuterus becomes reddish. Subsequently, PMSG, hMG or FSH is administeredfor 9 days. After confirming sufficient development of the ovarianfollicles, 400 IU/kg of human chorionic gonadotropin (hCG) is onceadministered intramuscularly. Thirty-eight to forty-two hours after theadministration of hGC, ovum collection is carried out. The ovumcollection is carried out by puncturing the ovarian follicles, andaspirating to collect ova together with follicular fluid using a 2.5 mlinjection syringe equipped with a 60 mm 19 G or 20 G Cathelin needle,wherein the syringe contains α-MEM solution containing about 0.5 ml of10% SSS (serum substitute supplement), under laparoscopic observation(outer diameter: 10 mm). Immediately after the collection, mature ovaeach enveloped by cumulus oophorus cells are separated under astereoscopic microscope, and transferred into TALP solution containing0.3% BSA. The ovum used for fertilization can be obtained by precultureof the above mature ova for 3 to 4 hours under the conditions at 37° C.in an atmosphere consisting of 5% CO₂, 5% O₂ and 90% N₂.

[0046] Also, a monkey sperm may be obtained from the epididymis, orobtained by an electric stimulation. The electric stimulation includesthe rectal method and the penile method, each described in Examplesbelow, and the like. Specifically, these methods are as follows:

[0047] Rectal Method

[0048] A male monkey is anesthetized with an anesthetic as representedby ketamine hydrochloride, xylazine hydrochloride and the like, andplaced in the supine position. Keratin cream is applied to a rod-shapedrectal electrode connected to an electric stimulator, and the electrodeis gently inserted into the rectum of the monkey. The electricstimulator is set at an alternating voltage of 5 to 20V. The electricityis applied intermittently, thereby collecting sperms from the tip of thepenis.

[0049] Penile Method

[0050] The limbs of a male monkey are held on the front face of a cageunder no anesthesia, and placed at a position in which the penis iseasily supported. An electrode is set to the penis and connected with aclip to an electric stimulator. The electricity is appliedintermittently, thereby collecting sperms from the tip of the penis.

[0051] It is desired that the monkey sperm used in the step (a)described above is activated from the viewpoint of high fertilities. Thesperm can be activated, for instance, by treatment with an agent such ascaffeine, dbC-AMP, forskolin and pentoxyfylline. Among the agents, acombination of caffeine and dbC-AMP is suitable from the viewpoints ofactive mobility with forward mobility, and the survival rate. Inaddition, after the treatment of sperm with the above-mentioned agent, asperm with even higher fertilities may be obtained by the Swim upmethod. There can be exhibited excellent effects that a highfertilization rate can be obtained by activating the sperm, and thatfertilization can be highly efficiently achieved by intracytoplasmicsperm injection even in the case of using a sperm which is poor inmobility without the treatment.

[0052] It is desired that the amounts of caffeine and dbC-AMP used are10 μM to 1 mM per 1×10⁷ sperms from the viewpoint of activating themobility.

[0053] The above-mentioned Swim up method is a method comprisingcollecting sperms in a round-bottomed tube by centrifugation, addingthereto a medium (about 0.5 ml) containing caffeine and dbC-AMP,allowing the cells to stand in an incubator at 37° C. under 5% CO₂, andthe collecting sperms which have swum up after about 30 to 60 minutes.

[0054] The activation of a sperm can be evaluated by using as an indexthe presence of active mobility with forward mobility.

[0055] The monkey sperms can be activated, for instance, by the methoddescribed as follows:

[0056] Sperms are transferred to a tube together with a lyophilizer froma straw in which sperms obtained from the epididymis, or obtained by anelectric stimulation have been stored. To the sperms, 10 ml of a BSA/BWW(Biggers, Whitten and Wittinghams) solution containing 1 mM caffeine and1 mM dbC-AMP is then added. Therefore, the sperms are incubated for 30minutes in a CO₂ incubator at 37° C. under 5% CO₂, to achieve spermcapacitation. Subsequently, the resulting products are centrifuged at1,000 rpm (200×g) for 2 minutes, to discard supernatant. To theremaining sperms, about 0.5 to 10 ml of a fresh BSA/BWW solutioncontaining 1 mM caffeine and 1 mM dbC-AMP is added. The resulting spermsolution is allowed to stand for 60 minutes in a CO₂ incubator at 37° C.The sperms swum-up are collected, and the mobility and the number of thesperms are confirmed. Here, the mobility of the sperms is evaluated byusing as indices forward mobility and activeness of the sperms. By thismethod, sperms can be activated.

[0057] In the step (a), fertilization can be carried out by in vitrofertilization or intracytoplasmic sperm injection. In vitrofertilization can be carried out according to the method described inTorii, R. et al., [Primates, 41, 39-47 (2000)]. In addition,intracytoplasmic sperm injection can be carried out according to themethod described in Hewitson, L. [Human Reproduction, 13, 3449-3455(1998)].

[0058] In the method for producing the monkey-derived embryonic stemcell of the present invention, it is preferable that a culture mediumselected from the group consisting of TALP(Tyrode-Albumin-Lactate-Pyruvate) solution, TALP-HEPES solution and BWWsolution is used for performing the in vitro fertilization orintracytoplasmic sperm injection mentioned above, from the viewpoint ofreducing the influence on the ovum. TALP solution and TALP-HEPESsolution can be prepared as follows: TABLE 1 Stock Solution Final StockSolution (ml) Reagent (mM) (g/100 mol) Solution (mM) TALP TALP-HEPESHEPES — 10.0 — 240 ml NaCl 157.0 0.92 114.0 to 100 ml to 100 ml KCl166.0 1.24 3.16 1.9 1.9 CaCl₂ 120.0 1.76 2.0 1.7 1.7 MgCl₂.6H₂O 120.02.44 0.5 0.41 0.41 Sodium Lactate 150.0 — 10.0 6.7 6.7 Water — — — 7.1NaH₂PO₄.H₂O 20.5 — 0.35 1.7 1.7 Glucose 295.0 5.31 5.0 NaHCO₃ 167.0 1.4025.0 (TALP) 15.0 1.2 2.0 (TALP- HEPES)

[0059] The stock solutions are sterilized by autoclave and then stored.

[0060] Here, immediately prior to the preparation of TALP solution,sodium pyruvate 0.5 mM 0.0055 g (per 100 ml) gentamycin sulfate (10mg/ml) 50 μg/ml 50 μl BSA 3 mg/ml 0.3 g

[0061] are prepared, and each of the reagents obtained is sterilized byfiltration through a filter.

[0062] On the other hand, immediately prior to the preparation ofTALP/HEPES solution, sodium pyruvate 0.1 mM 0.0011 g (per 100 ml) BSA 3mg/ml 0.3 g

[0063] are prepared, and each of the reagents obtained are sterilized byfiltration through a filter.

[0064] Incidentally, when TALP-HEPES solution is prepared, 50 ml of NaCland Na-HEPES (N-2-hydroxyethylpiperazine-N′-2-ethane sulfonate), phenolred and penicillin G are previously dissolved. To the resultingsolution, defined amounts of the respective stock solutions are added,and the NaCl stock solution is finally added to make up a volume of 100ml. The pH of the resulting solution is then adjusted to pH 7.4 with 1 MNaOH. The stock solution of sodium lactate is prepared by mixing anundiluted solution (60% syrup) with water in a ratio of 1:35. Phenol redis added at a concentration of 1 mg/ml to the resulting mixture.Thereafter, the pH of the resulting solution is adjusted to pH 7.6 with1 M NaOH. Subsequently, the solution is sterilized by filtration. Thereagent obtained is storable at 4° C. for one week. Twenty-eightmilligrams of NaHPO₄.H₂O is dissolved in 10 ml of a solution of glucose,and sterilized by filtration. The solution obtained is storable at 4° C.for one week.

[0065] Next, the composition of BWW (Biggers, Whitten and Wittinghams)solution is shown in Table 2. TABLE 2 Reagent Amount * (mg) SodiumChloride 2,770 Potassium Chloride 178 KH₂PO₄ 81 Magnesium Sulfate 147NaHCO₃ 1,053 Sodium Pyruvate Salt 14 D(+)-Glucose (anhydrous) 500Penicillin G 31 Streptomycin 25 DL-Sodium Lactate 1,037 Calcium Lactate263 Phenol Red 1 mg Merk 1

[0066] The surface of the solutions used for in vitro fertilization orintracytoplasmic sperm injection can be covered with a mineral oil orthe like. As a result, there can be obtained effects that drying an ovumsolution or a sperm solution is avoided, and that changes intemperature, pH, concentrations of CO₂ and O₂ are avoided.

[0067] In addition, in the step (a), it is preferable that a culturemedium selected from the group consisting of TALP solution, TALP-HEPESsolution and BWW solution is used during fertilization from theviewpoint of reducing the influence on the ovum.

[0068] Whether fertilization is achieved or not can be evaluated byvisual observation under a phase-contrast inverted microscope by usingas an index the presence of two pronuclei.

[0069] An example of in vitro fertilization or intracytoplasmic sperminjection performed in the present invention is shown below.

[0070] In vitro Fertilization

[0071] One to five ova each enveloped by cumulus oophorus cells areplaced in a mineral oil-covered drop of a 50 μl BSA/BWW solution on aplastic dish. A suspension of sperms is then transferred into the dropsat 5.0×10⁵ to 1.0×10⁶ sperms/ml. The drops are covered with a mineraloil, and insemination is then carried out.

[0072] Intracytoplasmic Sperm Injection

[0073] (i) Preparation of Ovum

[0074] The oocytes collected are gathered in a spot of a 50 μl TALPsolution containing 0.3% BSA (BSA/TALP solution), which is covered witha mineral oil (Sigma Chem. Co.), and precultured for about 2 to 4 hoursunder the conditions of 37° C., 5% CO₂, 5% O₂ and 90% N₂.

[0075] The state of maturation of ovum can be evaluated by removing thecumulus oophorus cells by treatment with 0.1% hyaluronidase, to therebycollect an ovum, and classifying the resulting ovum into the following 4classes, Class-1 to 4, under an inverted microscope.

[0076] Class-1: Mature ovum with a polar body (PB)

[0077] Class-2: Ovum being on maturation in which no PB and the germinalvesicle (GV) is observed

[0078] Class-3: Immature ovum in which the GV is observed

[0079] Class-4: Ovum with considerably deformed shape, or ovum showingdenaturation or retrogressive changes in the cytoplasm

[0080] An ovum classified in Class-1 is subjected to intracytoplasmicsperm injection immediately after the evaluation. An ovum classified inClass-2 or Class-3 is further gathered in a spot of a 50 μl BSA/TALPsolution, which is covered with a mineral oil, and continued to becultured under the conditions of 37° C., 5% CO₂, 5% O₂ and 90% N₂. Inthis case, the state of maturation of the ova is confirmed in the samemanner as in the above. It is desired that an immature ovum and ovumclassified in Class-4 are not used for fertilization.

[0081] (ii) Preparation of Sperms

[0082] The sperms are prepared in the same manner as in the in vitroFertilization.

[0083] (iii) Intracytoplasmic Sperm Injection

[0084] The intracytoplasmic sperm injection is carried out under aninverted microscope equipped with a micromanipulator.

[0085] Spot 1: 15 μl of a dilution of sperms, Spot 2: 3 spots of a 5 μl10% polyvinylpyrrolidone PBS culture medium [PVP: average molecularweight: about 360,000], and Spot 3: 3 spots of a 5 μl TALP-HEPESsolution (final concentration: 3 mg/ml BSA) for manipulation of ovum aresequentially placed in a dish of 150 mm in diameter, and thereaftertheir surfaces are covered with a mineral oil to avoid being dried. Thespots are used for the working field for intracytoplasmic sperminjection. Incidentally, when the temperature for manipulation is keptconstant, a heating stage may be used, if necessary.

[0086] An injection needle is connected to an ALCATHEL syringe of highlyworking performance. The injection needle includes a 30-degree inclinedneedle for human intracytoplasmic sperm injection.

[0087] An ovum supporting needle includes the above-mentioned 30-degreeinclined needle for human intracytoplasmic sperm injection, and a needlehaving an outer diameter of about 100 μm and an inner diameter of thetip of about 15 μm, which is made of a magnetic plur (PN-30,manufactured by Narishige). The ovum supporting needle is used in thestate where the needle is connected to an injector equipped with a2000-μl airtight syringe.

[0088] Regarding the sperm, a sperm having mobility selected to beaspirated in Spot 1 on the basis of the criteria for humanintracytoplasmic sperm injection, and thereafter the sperm obtained istransferred and discharged to Spot 2. In Spot 2, the mobility of thesperm is lowered due to the viscosity of PVP. The tail of sperm wasrubbed with the needle to partially destroy the membrane, thus, stoppingsperm movement. The sperm is aspirated together with a highly viscoussolution, and transferred to Spot 3.

[0089] The mature ovum is placed in Spot 3, and immobilized at a6-o'clock or 12-o'clock position in a clock using a supporting needle,while paying attention to avoid the destruction of the chromosome underthe polar body by the injection needle. Subsequently, the sperm isplaced in the tip of the injection needle, and injected into an ovum.After confirming that the needle is passed through the zona pellucida,the oolemma is aspirated. After confirming the rupture of the membrane,the contents (the sperms and the oosome) in the injection needle areinjected. A series of the manipulations for injecting a sperm and oosomeare repeated. Two or three ova are subjected to intracytoplasmic sperminjection in a single manipulation. When the inner surface of the tip ofthe needle is contaminated with sperms and oosome, the surface is washedwith Spot 2.

[0090] After the step (a), step (b) of allowing the fertilized ovumobtained in the step (a) to differentiate into a blastocyst by in vitroculture is carried out.

[0091] The in vitro culture includes a micro-hanging drop culturecharacterized in that a culture medium is covered with a mineral oilfrom the viewpoint of avoiding drastic changes in temperature and carbondioxide gas concentration. The micro-hanging drop culture is a techniquewhich is not usually employed in experiments for humans, but widelyemployed in the experiment for laboratory animals such as mouse andrabbit. According to this culture method, there can be exhibited anexcellent effect that an unexpectedly high development rate by applyingthe method in the development of a monkey-derived blastocyst.

[0092] For culturing a fertilized ovum, it is desired that the door ofan incubator should not be opened and the incubator is tightly closedfor 7 to 9 days, preferably 8 days after starting the cultivation in thecase of in vitro fertilization, and 7 to 10 days, preferably 9 daysafter starting the cultivation in the case of intracytoplasmic sperminjection, until the appearance of blastocyst is expected, from theviewpoint of avoiding unwanted stress, such as changes in temperatureand pH which can be caused during observation of the culture process.

[0093] The appearance of blastocyst tends to be proportional to the rateof early stage of cell division.

[0094] In addition, one of the significant features resides in culturemedium, culture temperature and culture atmosphere which are utilized inthe in vitro culture in the step (b).

[0095] It is preferable in the step (b) that the in vitro culture isperformed by using CMRL-1066, TCM-199, DMEM, α-MEM, or the like. It isespecially preferable that in vitro culture is performed usingCMRL-1066. Incidentally, CMRL-1066 can be prepared as follows:

[0096] The amount 0.014615 g (1 mM) of L-glutamine is dissolved in 10 mlof Solution A [penicillin G (1000 units), 0.5 ml of gentamycin sulfate(10 mg/ml), 10 ml of CMRL-1066 (10×) (without NaHCO₃ and L-glutamine),0.218 g of NaHCO₃, 6.7 ml of sodium lactate (290 mOsmol's stock), addedwith water to a volume of 100 ml]. The resulting solution is thensterilized by filtration. To 1 ml of the solution after thesterilization is added 9 ml of Solution A, to give Solution B in a totalvolume of 10 ml. The amount 0.0055 g (final concentration: 5 mM) ofsodium pyruvate is added to and dissolved in Solution B, to giveSolution C. Eight milliliters of Solution C and 2 ml of BCS (bovine calfserum) are mixed. The resulting mixture is sterilized by filtration, togive a solution of CMRL-1066.

[0097] Further, as the culture medium, it is desired that a culturemedium selected from the group consisting of TALP solution, TALP-HEPESsolution and BWW solution is used from the viewpoint of reducing theinfluence on the ovum. Specifically, the culture medium mentioned aboveincludes a culture medium in which TALP and CMRL-1066 are used incombination. According to the culture medium in which TALP and CMRL-1066are used in combination, there are some advantages in that by using theculture medium after confirmation of the fertilization allows the morulato develop into blastocyst, and to achieve extremely high developmentalratio of 40 to 46% in fertilized embryo.

[0098] It is desired that the culture temperature is 37° C. or higher,preferably 37.5° C. or higher, and that the culture temperature is 38.5°C. or lower, preferably 38.2° C. or lower, from the viewpoint ofshortening the time required in the development, and the viewpoint ofpromoting of development from morula into blastocyst. Specifically, at 7days after in vitro fertilization, and at 8 days after intracytoplasmicsperm injection, blastocysts can be efficiently obtained by culturing at38° C.

[0099] The culture atmosphere is preferably a low-oxygen atmosphere fromthe viewpoint of promoting or improving the development from morula toblastocyst. Specifically, by using an atmosphere consisting of 5% CO₂,5% O₂ and 90% N₂, which is lower in O₂ concentration than a cultureatmosphere usually employed for preparation of ES cells, there can beexhibited excellent effects such that blastocysts can be obtainedsurprisingly efficiently.

[0100] Further, step (c) of establishing an embryonic stem cell lineusing the blastocyst obtained in the step (b) is carried out. In thestep (c), the embryonic stem cell line is established by culturing theinner cell mass derived from the blastocyst obtained in the step (b) onfeeder cells or in the presence of leukemia inhibitory factor [LIF, alsoreferred to as differentiation inhibitory factor (DIF)].

[0101] When inner cell mass is obtained from a blastocyst, blastocystsin which the zona pellucida has been removed may be used. The zonapellucida may also be removed by treating with hyaluronidaze, pronase,acidic Tyrode's solution and the like. When the zona pellucida isremoved by hyaluronidaze, pronase, acidic Tyrode's solution and thelike, blastocysts may be incubated in M2 culture medium containing anappropriate concentration of hyaluronidaze, pronase, acidic Tyrode'ssolution or the like [see, e.g., D. M. Glover et al., ed., DNA Cloning4, Mammalian Systems, A Practical Approach 2nd edition (1995), and thelike]. After removing the zona pellucida, the blastocysts obtained maybe washed with phosphate-buffered saline, if necessary.

[0102] In order to separate the inner cell mass from a blastocystwithout the zona pellucida, for instance, the blastocyst may besubjected to immunosurgery. Further, endodermal cells are stripped awayby pipetting. The resulting inner cell mass obtained is cultured onfeeder cells for 1 week. The proliferated inner cell mass is trypsinized(for instance, treated with 0.25% by weight of trypsin+0.5 mM EDTA, andthe like), to obtain a mass made of about 3 to 4 cells. Further, thesecells are cultured again on feeder cells.

[0103] The anti-serum used in the immunosurgery includes, for example, arabbit anti-monkey serum, particularly a rabbit anti-Japanese monkeyserum, a rabbit anti-cynomolgus monkey serum and the like. The innercell mass can be separated by transferring the blastocysts into asolution prepared by 20-fold diluting the anti-serum with M16 culturemedium [see DNA Cloning 4, Mammalian Systems, A Practical Approach,supra, and the like], and incubating them at 37° C. for 30 minutes. Inaddition, the trophectoderm may be physically removed under a microscopeusing a glass needle, as required.

[0104] The feeder cell includes a cell obtained by treating withmitomycin C or irradiating with X ray, and the like a fibroblasticprimary cell culture derived from a murine fetus at Day 12 to Day 16from pregnancy, murine fetal fibroblast cell line, STO cell, and thelike. The mouse-derived feeder cells are advantageous for experiments inthat they can be prepared in a large scale.

[0105] The preparation of a feeder cell can be carried out, forinstance, by the method described in Examples below, and the like.

[0106] The feeder cells are plated to a gelatin-coated culture vesselusing MEM (minimum essential medium eagle) medium. The feeder cells maybe plated on a culture plate to an extent that the cells cover thesurface of the plate without spacing.

[0107] The inner cell mass is plated on feeder cells after exchangingMEM medium with a medium for culturing ES cells [ES cell culture medium,Table 3], in a culture vessel in which the feeder cells are plated,.TABLE 3 Composition of ES Cell Culture Medium Name of ManufacturalProduct Amount Added DMEM/F12 (manufactured by Sigma) 500 ml FBS(manufactured by JRH BIOSCIENCES) 75 ml Glutamine (manufactured bySigma; 200 mM) 5 ml Mixture of Penicillin (manufactured by 5 ml Sigma;10,000 IU/ml) and Streptomycin (manufactured by Sigma; 10 mg/ml) SodiumPyruvate (manufactured 5 ml by Sigma; 100 mM) Sodium Hydrogencarbonate(manufactured 8 ml by Sigma; 7.5%) 2-Mercaptoethanol 4 μl (manufacturedby Sigma; final concentration: 10⁻⁴ M) LIF 0.5 ml of one having(manufactured by ESGRO; final 10⁶ U/ml concentration: 1000 U/ml)

[0108] The culture conditions for the cells may be the conditionsutilized usually for a culture condition of mouse ES cells. Forinstance, the cells may be cultured for 7 days under the conditions at37° C. in 5% CO₂. Incidentally, it is desired that the culture medium isnot exchanged for 3 days after starting the culture and that the stateof implantation is observed every day under a microscope, from theviewpoint of avoiding the prevention of implantation of inner cell mass.In addition, the cells may be sequentially transferred to a larger platefor subculture as the cells proliferate.

[0109] Next, the embryonic stem cells are identified and evaluated.Incidentally, the exemplary evaluation criteria are as follows.

EXAMPLES OF EVALUATION CRITERIA

[0110] Karyotype

[0111] The chromosome number is not abnormal. Typically, whether or notthe chromosome number is the same as that of the original monkey (2n=42)is determined.

[0112] Pluripotency

[0113] For instance, the potential to differentiate can be examined byinjecting cells (1×10⁵ to 1×10⁶ cells), which are appeared to beembryonic stem cells, to hypodermis, the renal capsule or in the testisof a 8-12 week old SCID mouse or nude mouse, examining the presence orabsence of the tumor formation at 5 to 16 weeks after the injection,wherein when tumors are formed, the tumors are subjected to thehistological test. Also, various types of potentials to differentiatecan be determined and the pluripotency can be evaluated by removing thefeeder cells or by adding a differentiation-inducing agent such asretinoic acid.

[0114] Morphological Features p1 1. There are exhibited highnucleus/cytoplasm ratio, conspicuous nucleoli, and colonization.

[0115] 2. The colonies are more flat, as compared to the mouse ES cellcolonies.

[0116] Examples of the morphological features are shown in Panel A andPanel B in FIG. 1 and FIG. 2.

[0117] Expression of Cell Surface Markers

[0118] Negative Control: SSEA-1

[0119] Positive Control: SSEA-3, SSEA-4

[0120] The respective cell surface markers above are glycolipid cellsurface markers which are stage-specific embryonic antigens. The markerscan be detected by a conventional immunostaining or the like usingantibodies raised against these markers as antigens.

[0121] Alkaline Phosphatase Activity

[0122] The alkaline phosphatase activity can be detected by aconventional alkaline phosphatase staining.

[0123] Specifically, the monkey-derived embryonic stem cell of thepresent invention includes an established cynomolgus monkey-derivedcell, specifically an embryonic stem cell, having the followingproperties:

[0124] (i) being capable of indefinitely proliferating while beingmaintained in an undifferentiated in state;

[0125] (ii) having the same chromosome number as that of original Macacafascicularis;

[0126] (iii) exhibiting the pluripotency when transplanted tohypodermis, the renal capsule or the testis of a 8-12 week-old SCIDmouse or nude mouse;

[0127] (iv) being negative for SSEA-1 and positive for SSEA-3 andSSEA-4; and

[0128] (v) exhibiting an alkaline phosphatase activity.

[0129] The cynomolgus monkey-derived embryonic stem cell has thefollowing morphological features: (vi) exhibiting high nucleus/cytoplasmratio, conspicuous nucleoli, and colonization, and being more flatcolonies compared to the mouse ES cell colonies (see, for example, PanelA and Panel B in FIG. 1 and FIG. 2).

[0130] The established cynomolgus monkey-derived embryonic stem cell ofthe present invention exhibits the ability to differentiate intoectodermal cells, mesodermal cells, endodermal cells and the like, morespecifically the ability to differentiate into, for example, neuron,glia, muscle, cartilage, bone, ciliated epithelium, intestinalepithelium and the like, when transplanted to hypodermis, the renalcapsule or the testis of a 8-12 week-old SCID mouse or nude mouse.

[0131] The established cynomolgus monkey-derived embryonic stem cell ofthe present invention is expected to be used for generating modelanimals of diseases and for preparing tissues for transplantation.

[0132] The monkey-derived embryonic stem cell of the present inventionallows the screening of a reagent for specific differentiation into atissue or a cell, especially preferably of a primate tissue or a primatecell. The present invention encompasses a method for screening a reagentfor specific differentiation into a tissue or a cell.

[0133] One of the features of the method for screening a reagent forspecific differentiation into a cell or tissue of the present inventionresides in that the embryonic stem cell of the present invention is keptin the presence of a test substance.

[0134] In the screening method of the present invention, since theembryonic stem cell of the present invention is used, there areexhibited excellent effects that the reagent useful for embryologicalstudies, studies of diseases and clinical applications and useful forobtaining the desired differentiated cells or differentiated tissues canbe screened.

[0135] In the screening method of the present invention, the specificdifferentiation from an embryonic stem cell into the desired tissue orcell can be evaluated by, for example, using as an index a markerexpressed in the desired tissue or cell. The marker for the desiredtissue or cell includes tissue- or cell-specific antigens. The markerfor the desired tissue or cell includes, for example, neuron-specificenolases, glia fibrous acidic proteins, nestin and the like as a markerfor a neuronal cell; S-100 protein, tartaric acid-resistant acidphosphatase and the like as a marker for cartilage; desmin,muscle-specific actin and the like as a marker for muscle. Such specificmarker may be detected by conventional ELISA, immunostaining or the likeusing antibodies against the markers, or detected by conventionalRT-PCR, DNA array hybridization or the like using a nucleic acidencoding the marker. Incidentally, the term “nucleic acid” refers togenomic DNA, RNA, mRNA or cDNA.

[0136] The reagent obtained by the screening method is encompassed bythe present invention. According to the reagent, applications in thefield of regenerative medicine are expected.

[0137] In addition, the differentiated cell and differentiated tissueeach differentiated from the embryonic stem cells of the presentinvention are also encompassed by the present invention.

[0138] The differentiated cell and differentiated tissue can beidentified by observing the expression of the markers specific for thecell or tissue, or the morphological features.

[0139] The differentiated cell or differentiated tissue of the presentinvention is a cell or tissue of monkey which is closely related tohumans. Therefore, the cell or tissue is suitably used as a subject in avariety of tests for various agents, or to use as a model fortransplantation of tissue or cell.

[0140] The present invention will be more specifically described belowby the following Examples and the like, but is not limited to theExamples. Here, “%” as used in the Examples and the like indicates % byweight, unless otherwise specified. Also, the expression “%” for CO₂, O₂and N₂ indicates % by volume.

Example 1

[0141] Method for Preparing Blastocyst from Cynomolgus Monkey

[0142] In a monkey, unlike in other laboratory animals such as a mouse,a rat and a rabbit, a method for collecting a fertilized ovum byperfusion of an oviduct or uterus remains yet to be established in thecurrent situation. It has been also known that the efficiency of amethod for collecting a fertilized ovum from the uterus in the ovulationcycle is very low.

[0143] Therefore, at this time, in order to produce a blastocystsuitable for the establishment of an embryonic stem cell, there wasinvestigated a method comprising carrying out fertilization by in vitrofertilization and intracytoplasmic sperm injection, and developing to ablastocyst by in vitro culture.

[0144] (1) Ovary Stimulation Method

[0145] To a female cynomolgus monkey (4-15 year-old), 1.8 mg ofgonadotropin-releasing hormone (GnRH) [trade name: Leuplin, manufacturedby Takeda Chemical Industries, Ltd.; or trade name: Sprecur,manufactured by Hoechst Marion Roussel] was subcutaneously administered.Starting at 2 weeks after GnRH administration, 25 IU/kg pregnant mare'sserum gonadotropin (PMSG) [trade name: Serotropin, manufactured byTeikoku Hormone Manufacturing, Co., Ltd.], 10 IU/kg human menopausalgonadotropin (hMG) [trade name: Pergonal, manufactured by TeikokuHormone Manufacturing, Co., Ltd.] or 3 IU/kg follicle-stimulatinghormone (FSH) [trade name: Fertinorm, manufactured by SeronoLaboratories] was intramuscularly administered at a given time in theevening once a day for nine consecutive days, in this Example. Five daysafter the administration, the ovary was observed by using a laparoscope(3 mm in outer diameter), thereby confirming the presence or absence offollicle development.

[0146] Subsequently, after confirming sufficient follicle development byadministration of PMSG, hMG or FSH, 400 IU/kg human chorionicgonadotropin (hCG) [trade name: Puberogen, manufactured by Sankyo Co.,Ltd.] was administered once intramuscularly. Forty hours after hCGadministration, ova were collected.

[0147] The ovum collection was carried out by puncturing the ovarianfollicles, and aspirating to collect ova together with follicular fluidunder a laparoscope observation (10 mm in outer diameter) using a 2.5 mlinjection syringe equipped with a 60 mm 19G or 20G Cathelin needle,wherein the syringe contains a solution of α-MEM (alpha-modification ofEagles Medium, manufactured by ICD Biomedical Inc.) containing about 0.5ml of 10% SSS (serum substitute supplement, manufactured by IrvineScientific Sales Inc.).

[0148] After the collection, a mature ovum enveloped by cumulus oophoruscells was immediately separated under a stereoscopic microscope,transferred to TALP containing 0.3% BSA (hereinafter referred to asBSA/TALP), and pre-cultured for 3 to 4 hours in a carbon dioxideincubator at 37° C., 5% CO₂, 5% O₂ and 90% N₂.

[0149] As described above, the ovum collection under laproscopicobservation was carried out by making only about 1 cm incision in theabdominal wall, inserting a laparoscope and puncturing the ovary throughthe abdominal wall.

[0150] On the other hand, the ovum collection method was performed bythe ovarian extirpation method comprising extirpating ovary and uterus,and thereafter perfusing uterine tube, in the case of a mouse, or by theovarian puncture method using ultrasonic diagnostic equipment, in thecase of human. In the case of a monkey, however, the ovarian extirpationmethod is inapplicable in some cases because of the limited number ofindividuals. In addition, since a monkey is smaller in body size thanhuman, it is extremely difficult to apply the method using ultrasonicdiagnostic equipment. For these reasons, a method for collecting anunfertilized ovum for in vitro fertilization of a monkey, both atdomestically and abroad, are usually performed by the ovarian puncturemethod under direct vision of the ovary by laparotomy. However, thismethod has drawbacks such as {circle over (1)} larger burden on anindividual, {circle over (2)} much time consumption for postoperativewound healing, and {circle over (3)} major risk of infection.

[0151] In contrast, the method employed in the present Example provideseven magnified views compared to the case of the direct vision. It istherefore possible to collect ova with the puncture site targetedaccurately. In addition, since the operation can be completed in a veryshort time by suturing a single stitch for the abdominal wall after ovumcollection, it was found that the present method is very useful from theviewpoint of animal welfare.

[0152] (2) Collection of Sperm

[0153] (i) Collection Method from Epididymis

[0154] Immediately after collecting the epididymis of a male cynomolgusmonkey (10-15 year-old), a 1 ml injection syringe equipped with a 23Gneedle was inserted to the seminal duct. Thereafter, BWW containing 0.3%BSA (hereinafter referred to as BSA/BWW) was injected slowly, therebycollecting the semen overflowed from the tail of the epididymis cut.

[0155] (ii) Collection Method by Electric Stimulation

[0156] i) Rectal Method

[0157] A male cynomolgus monkey (10-15 year-old) was anesthetized with acombination of ketamine hydrochloride and xylazine hydrochloride (5mg/kg and 1 mg/kg, respectively) and placed in the supine position.Keratin cream was applied to a rod-shaped rectal electrode connected toan electric stimulator. Thereafter, the electrode was gently inserted tothe rectum of the monkey. The penis was washed with sterilephysiological saline and wiped with paper towel or the like. The penistip was immersed in a test tube (50 ml). Subsequently, the electricitywas conducted by setting the electric stimulator to an alternatingvoltage of 5 V. The electricity was conducted for 3 to 5 seconds andthen paused for 5 seconds. This procedure was repeated in up to threecycles. If ejaculation was observed, stimulation was terminated at thattime. If ejaculation was not observed, the same procedure was performedby changing the voltage to 10 V. If ejaculation was still not observed,the procedure was carried out at 15 V and 20 V.

[0158] ii) Penile Method

[0159] Under no anesthesia, the four limbs of a male cynomolgus monkey(10-15 years old) were held on the front face of a cage and placed at aposition that the penis is easily supported. The operator, wearingsurgical rubber gloves, washed the monkey's penis with sterilephysiological saline and wiped with paper towel or the like. An electricstimulator was provided and thereafter, an electrode was set to thepenis and connected with a clip to the stimulator. The electricity wasconducted by repeating the procedure of ON-OFF at 1-second intervals ata direct current voltage of 5 V while shortening the interval graduallywith the passage of time. If ejaculation was not observed, the sameprocedure was performed at a voltage of 10 V, 15 V, and 20 V. Ifejaculation was still not observed, the same procedure was performedwith an alternating current.

[0160] (3) Treatment after Semen Collection and Cryopreservation (Toriiet al., 1998)

[0161] The semen collected by the rectal method or the penile method wasallowed to stand for about 30 minutes in a carbon dioxide incubator at37° C. A sperm solution was prepared by collecting only a liquidcomponent, and adding thereto about 1 to 2 ml of BWW containing 0.3% BSA(Biggers, Whitten and Wittinghams) (BSA/BWW). Thereafter, the resultingsolution was gently overlaid on a layered liquid of 2.5 ml of 80%Percoll (manufactured by American Permacia Biotech Inc.) and 2.5 ml of60% Percoll. After centrifugation of the resulting product at 1,400 rpmand at room temperature for 20 minutes, the upper layer was aspiratedoff with about 0.5 ml of the bottom portion remaining in the test tube.Further, about 10 ml of BSA/BWW was added thereto and mixed gently.After centrifugation of the mixture at 1,400 rpm and at room temperaturefor 3 minutes, the upper layer was aspirated off with about 0.5 ml ofthe bottom portion left in the test tube.

[0162] An appropriate amount of BSA/BWW was added to the sperm fractionso as to reach a sperm count of about 5×10⁷ to 1.0×10⁸ cells/ml, toprepare a sperm solution. Thereafter, the resulting solution was allowedto stand at 4° C. for about 60 to 90 minutes. Subsequently, to thesolution, a TTE-G solution [TTE medium (composition per 100 ml of themedium: 1.2 g Tes, 0.2 g Tris-HCl, 2 g glucose, 2 g lactose, 0.2 graffinose, 20 ml egg yolk, 10,000 IU penicillin-G, 5 mg streptomycinsulfate) containing glycerol at a final concentration of 12%], in aone-fifth amount of the amount of the sperm solution, was gently addeddrop by drop in ice water, and thereafter, the resulting mixture wasallowed to stand for 5 minutes. The above-mentioned procedures ofdropping of TTE-G solution and allowing to stand were repeated in fivecycles.

[0163] After allowing the resulting mixture to stand for 60 to 90minutes in ice water, the sperm solution was placed in a 0.25 or 0.5 mlstraw. The straw was kept above a vessel containing liquid nitrogen forabout 5 minutes, and thereafter, it was further kept above the liquidnitrogen for 5 minutes. The straw was placed in the liquid nitrogen andstored.

[0164] (4) Preparation of Sperm for In vitro Fertilization

[0165] The straw was taken out from the liquid nitrogen, kept at roomtemperature for 30 seconds, and then immersed in a 37° C. hot bath for30 seconds, to thereby thaw the stock sperm solution. Subsequently, thecapacitation was carried out by adding 10 ml of BSA/BWW containing 1 mMcaffeine (manufactured by Sigma Ltd.) and 1 mM dbC-AMP (manufactured bySigma Ltd.) to the straw, and incubating the resulting mixture in acarbon dioxide incubator at 37° C. for 30 minutes.

[0166] Thereafter, the sperm solution was centrifuged at 1,000 rpm (200g) for 2 minutes, to discard the supernatant. Subsequently, about 0.5 to1 ml of BSA/BWW containing 1 mM caffeine and 1 mM dbC-AMP was added tothe sperms. The sperm solution obtained was allowed to stand for 60minutes in a carbon dioxide incubator at 37° C. Sperms that were swum upwere collected, and confirmed for their mobility and number. Sperms forin vitro fertilization were thus obtained.

[0167] (5) Method of Fertilization

[0168] 1) In vitro Fertilization

[0169] One to five ova each enveloped by cumulus oophorus cells wereplaced in a drop of a 50 μl BSA/BWW covered with mineral oil on aplastic dish. Subsequently, the sperm suspension was transferred to thedrop to obtain a sperm density of 5.0×10⁵ to 1.0×10⁶ sperms/ml. Aftercovering the drop with mineral oil, insemination was then carried out.

[0170] The fertilized ova were then incubated in a carbon dioxideincubator at 37° C., 5% CO₂, 5% O₂ and 90% N₂. Five hours afterinsemination, the BWW solution was exchanged with TALP solution, andthereafter, the ova were examined for fertilization. As a result,fertilized ova were obtained at a fertilization rate of as high as about45%. The ova confirmed to have fertilized were cultured for about 20hours, transferred to CMRL-1066 solution and continued to be cultured.

[0171] 2) Intracytoplasmic Sperm Injection

[0172] (i) Preparation of Ova

[0173] The collected oocytes were gathered in a 50 μl spot of a solutionof TALP containing 0.3% BSA (BSA/TALP) covered with mineral oil(manufactured by Sigma Ltd.), and then pre-cultured for about 2 to 4hours at 37° C. in the presence of 5% CO₂, 5% O₂ and 90% N₂.

[0174] To confirm the state of maturation of the ovum, the oocyteculture was exposed to a TALP-HEPES solution containing 0.1%hyarulonidase (manufactured by Sigma Ltd.) for 1 minute, and thereaftercumulus oorphorus cells were removed by pipetting. The recovered ovawere classified under an inverted microscope into classes 1 through 4 asfollows:

[0175] Class-1: Mature ovum with polar body (PB)

[0176] Class-2: Ovum being on maturation in which no PB and germinalvesicle (GV) are observed

[0177] Class-3: Immature ovum in which GV is observed

[0178] Class-4: Ovum with considerable deformed shape, or ovum showingdenaturation or retrogressive changes in the cytoplasm.

[0179] The ovum classified in class-1 was subjected to intracytoplasmicsperm injection immediately after class confirmation. The ova classifiedin class-1 or class-3 were further gathered in a 50 μl spot of aBSA/TALP solution, which was covered with mineral oil, and then culturedat 37° C., 5% CO₂, 5% O₂ and 90% N₂. Twenty-four hours aftercultivation, the state of maturation of the ovum was confirmed. Themature ovum was subjected to intracytoplasmic sperm injection at thattime. The remaining immature ova and the ova each classified in class-4were not subjected to fertilization.

[0180] (ii) Preparation of Sperm

[0181] Sperms were prepared in the same manner as in vitrofertilization.

[0182] (iii) Intracytoplasmic Sperm Injection

[0183] Intracytoplasmic sperm injection was carried out under an OlympusIX 70 inverted microscope which was equipped with a micro-manipulatormanufactured by Narishige Co., Ltd.

[0184] Spot 1: 15 μl of dilution of sperms, Spot 2: 3 spots of a 5 μl10% polyvinyl pyrrolidone PBS culture medium [PVP: average molecularweight ca. 360,000, manufactured by nacalaitesque], and Spot 3: 3 spotsof a 5 μl solution of TALP-HEPES for ovum manipulation (finalconcentration 3 mg/ml BSA) were sequentially placed in a 150 mm dish.Thereafter, their surfaces were covered with mineral oil to avoid frombeing dried. This dish was used as the working field forintracytoplasmic sperm injection. In the present Example, no attentionwas paid to a change in operating temperature, and no heating stage wasused.

[0185] As the injection needle, there was used a 30 degree inclinedneedle for human intracytoplasmic sperm injection (7-8 μm in outerdiameter and 5-7 μm in inside diameter, manufactured by MediconInternational). The needle was connected to an ALCATEL syringe of highworking performance.

[0186] As the ovum supporting needle, there was used the same 30 degreeinclined needle for human intracytoplasmic sperm injection or a needleabout 100 μm in outer diameter and about 15 μm in tip inside diameterprepared with a magnetic plur (trade name: PN-30, manufactured byNarishige Co., Ltd.). This needle was connected to a Narishige injectorequipped with a 2000 μl airtight syringe.

[0187] In Spot 1, a sperm having mobility was selected and aspirated onthe basis of the Human Intracytoplasmic Sperm Injection Standard. Theresulting sperm was then transferred to Spot 2 and discharged. In Spot2, the mobility of sperm was lowered due to the viscosity of PVP. Thetail of the sperm was rubbed with the injection needle to partiallydestroy the membrane, thus stopping sperm movement. The sperm wasaspirated together with the highly viscous solution and transferred toSpot 3.

[0188] A mature ovum was placed in Spot 3 and immobilized at a 6-o'clockor 12-o'clock position using the ovum supporting needle while payingattention to avoid the destruction of the chromosome under the polarbody by the injection needle. Subsequently, the sperm was placed in thetip of the injection needle and injected into the ovum. After confirmingneedle passage through the zona pellucida, the oolemma was aspirated.After confirming the rupture of the membrane, the contents (sperm andovum cytoplasm) in the injection needle were injected. A series of theseprocedures for injection of sperms and ovum cytoplasm were repeated. Twoor three ova are subjected to intracytoplasmic sperm injection in asingle manipulation. When the inside of the tip is contaminated withsperms or oosome, the tip is washed with spot 2.

[0189] The ova resulting from intracytoplasmic sperm injected werereturned to the incubator. The cultivation was then started at 37° C.,5% O₂, 5% CO₂ and 90% N₂. Immediately after intracytoplasmic sperminjection, a 50 μl spot of a CMRL-1066 solution was made on a 60 mmnon-coated culture dish and covered with paraffin oil. As a generalrule, equilibration between the spot and the gas phase was carried outfor at least 3 hours. Twenty-four hours after intracytoplasmic sperminjection, the ovum was transferred from the TALP solution to theabove-mentioned spot of CMRL-1066 solution and cultured for 8 days in acarbon dioxide incubator at 37° C., 5% O₂, 5% CO₂ and 90% N₂ underclosed conditions. As a result, fertilized ova were obtained at afertilization rate of as high as about 75 to 85%.

[0190] As described above, the activation of sperms was carried outprior to in vitro fertilization and intracytoplasmic sperm injection inmonkey. In the cases of mouse and human, intact sperm for ordinary invitro fertilization and intracytoplasmic sperm injection are used.However, in the Example, a sperm having higher fertility was obtained bycarrying out activation with caffeine and dbC-AMP, and then employingSwim-up method, in order to achieve a higher fertilization rate. It wasfound that high fertilization can be achieved by employing thisadditional procedure in in vitro fertilization. Furthermore, the sametreatment resulted in a high fertilization rate in intracytoplasmicsperm injection even with spermatozoa unsuitable for in vitrofertilization due to a lack of mobility. It was found that this methodwas very effective in a case where sperm of poor fertilization rate hasbeen supplied.

[0191] (6) Method for Cultivation

[0192] In in vitro fertilization and intracytoplasmic sperm injection,the micro-hanging drop culture method by medium coverage with mineraloil, which is usually not employed in humans but widely employed inlaboratory animals such as a mouse and a rabbit, was used to avoid rapidchanges in temperature and carbon dioxide gas concentration duringcultivation after confirmation of fertilization. In addition, to avoidunwanted stress due to changes in temperature or pH associated withexamination of the course of cultivation, cultivation was carried outunder closed conditions until the time when appearance of blastocystswas expected without opening or closing of the incubator door for 7 daysafter cultivation initiation for in vitro fertilization and 8 days aftercultivation initiation for intracytoplasmic sperm injection.

[0193] The culture medium, cultivation temperature, and cultivation gasphase used here are as follows:

[0194] Culture Medium: TALP & CMRL-1066

[0195] When BWW, which is commonly used in the case of a mouse, PI(manufactured by Nakamedical, Inc.) or Blast medium (manufactured byNakamedical, Inc.), which are commonly used in human, or newly developedHFF (human follicular fluid, manufactured by Fuso PharmaceuticalIndustries, Ltd.), development progresses smoothly to the fertilizationand segmentation stages but stops at the morula stage. It was found thatby using in a combination of TALP culture medium and CMRL-1066 culturemedium, the blastocyst as found to be developed, and that thedevelopmental ratio was found to be an extremely high ratio of 40 to 46%in fertilized embryos. It is considered that the adverse effect on ovawas reduced by the use of TALP, which is derived from HEPES buffersolution, rather than PBS, a phosphate buffer solution, in theprocedures outside the incubator.

[0196] Culture Temperature: 38° C.

[0197] Embryo culture is usually carried out at 37° C. in a mouse andhuman. In a monkey, however, development at this temperature was slowwith no development to the morula stage and thereafter. Therefore,cultivation in the present Example was carried out at a slightly highertemperature of 38° C., as compared to the case of bovines and otheranimals whose embryos are cultured at 38.5° C. As a result, blastocystswere obtained at 7 days after cultivation for in vitro fertilization and8 days after cultivation for intracytoplasmic sperm injection.

[0198] Culture Atmosphere: 5% CO₂, 5% O₂ and 90% N₂

[0199] Although development stopped at the morula stage under thecommonly used conditions of 5% CO₂ and 95% air, a high ratio ofdevelopment into the blastocysts was observed by culturing fertilizedova at 5% CO₂, 5% O₂ and 90% N₂.

Example 2

[0200] Method for Establishing Monkey ES Cells

[0201] (1) Preparation of Feeder Cells

[0202] Primary embryonic fibroblasts (hereinafter also referred to asPEFs) obtained from a 12.5 day-old mouse embryo were cultured forprimary generation to third generation in an MEM medium containing 10%fetal bovine serum (FBS) until the cells became confluent. Subsequently,the PEFs were cultured for 2 to 3 hours in an MEM medium containingmitomycin C (MMC) at a final concentration of 10 μg/ml to inactivatecell division. The medium containing MMC was then removed. The cellswere washed three times with PBS. The washed cells were trypsinized(0.05% trypsin and 1 mM EDTA), and removed from the culture dish.Thereafter, the number of the cells were counted.

[0203] The MMC-treated PEFs were plated to a gelatin-coated 24-wellculture dish at 2×10⁴ cells per well.

[0204] After plating actually the resulting cells onto the dish andconfirming an appropriate number of the resulting cells, the mouse EScells were cultured and their nature was examined. As a result, sincethe ES cells had a good ability to proliferate and were maintained in anundifferentiated condition, the cells obtained were thus shown to besuitable as feeder cells. In addition, feeder cells obtained until thethird generation (primary generation to third generation) werepreferred.

[0205] (2) Separation of Inner Cell Mass from Monkey Blastocysts

[0206] To remove the zona pellucida, the monkey blastocysts weretransferred to an M2 culture medium [see for example D. M. Glover etal., ed., DNA Cloning 4 Mammalian Systems—A Practical Approach—, 2ndedition (1995), and the like] containing pronase or Tyrode at a finalconcentration of 0.5% and incubated at 37° C. for 10 minutes. Theblastocysts with remaining zona pellucida were further treated withpronase at 37° C. for 5 minutes. After confirming the removal of thezona pellucida, the blastocysts obtained were washed twice with PBS.

[0207] Subsequently, the blastocysts were transferred to a solutionprepared by 20-fold diluting rabbit anti-cynomolgus monkey lymphocyteserum with M16 culture medium [see above-mentioned DNA Cloning 4Mammalian Systems—A Practical Approach—and the like] and incubated at37° C. for 30 minutes. The blastocysts obtained were then washed threetimes with PBS. The blastocysts were transferred to a solution preparedby 50-fold diluting the complement with M16 culture medium, andincubated at 37° C. for 30 minutes. The blastocysts obtained were washedthree times with PBS. If the trophectoderm of blastocysts could not becompletely removed by these procedures, the trophectoderm was physicallyremoved using a glass needle under a microscope. Inner cell mass (ICM)was thus separated.

[0208] (3) Culture of Monkey Inner Cell Mass

[0209] The MEM medium was removed from the 24-well culture dish in whichthe feeder cells obtained in the item (1) were plated. Thereafter, 800μl of the culture medium for ES cells (Table 3) was added to each well.

[0210] Subsequently, the ICM obtained in the item (2) was transferred toeach of the wells at one cell per well using a micropipette and culturedat 37° C., 5% CO₂ for 7 days. The state of implantation was observedevery day under a microscope without exchanging the medium with a freshsupply for 3 days of cultivation in order to avoid preventing ICMimplantation.

[0211] On day 7 of cultivation, the ICM cells were macerated. Afterremoving the culture medium for ES cells from the wells, the dish wasonce washed with PBS. Three-hundred microliters of 0.25% trypsin/0.02%EDTA was added to each well and immediately removed. Subsequently, the24-well culture dish was incubated at 37° C. for 1 minute. Afterconfirming cell maceration under a microscope, 500 μl of the ES cellmedium was added to each well and thoroughly pipetted using PIPETMAN.

[0212] All the above cells were transferred to the wells of a 24-wellculture dish previously plated with the feeder cells. After adding 300μl of the ES cell medium to obtain a total medium volume of 800 μl, themedium was thoroughly mixed to avoid uneven cell plating. The ES cellmedium was exchanged with a fresh supply every two days. Since a groupof cells deemed ES cells proliferate to colonize within 7 days aftercell maceration, the medium was examined every day.

[0213] After a colony of ES cells appeared, the cells on the 24-wellculture dish were trypsinized and subcultured repeatedly. During thisoperation, the ES cell medium was replaced with a fresh supply every dayor every two days. As a result, more than one ES cell line was obtainedfrom cynomolgus monkey blastocysts.

Example 3

[0214] Evaluation of Monkey ES Cells

[0215] (1) Cynomolgus Monkey ES Cells

[0216] Karyotype:

[0217] Whether or not the chromosome number was normal (same as thechromosome number of the origin monkey: 2n=42) was determined. As aresult, the established ES cell lines were found to retain the normalkaryotype.

[0218] Pluripotency:

[0219] 1×10⁶ cynomolgus monkey ES cells were subcutaneously injected tothe inguinal region of an 8-week-old SCID mouse. Five to twelve weeksafter injection, tumor formation was observed. After fixation in Bouin'sfluid or a para-formaldehyde solution, the tumor was sectioned,subjected to hematoxylin-eosin staining (HE staining) or immunologicalstaining, and examined histologically. In the immunostaining, anantibody against human neuron-specific enolase (NSE), an antibodyagainst glia fibrous acidic protein (GFAP), an antibody against S-100protein and an antibody against desmin were used since the availabilityof monkey tissue-specific antibodies was limited.

[0220] As a result, the tumor formed was identified as a teratomaconsisting of a group of cells derived from ectoderm (neuron and glia),mesoderm (muscle, cartilage, and bone) and endoderm (ciliated epitheliumand intestinal epithelium). In the immunohistologic examination, neuronwas detected by the antibody against NSE, glia being detected by theantibodies against NSE and GFAP, peripheral nerves being detected by theantibody against NSE, cartilage being detected by the antibody againstS-100 protein, and muscles being detected by the antibody againstdesmin. These results showed that cynomolgus monkey ES cells have thepluripotency into ectoderm-derived cells, mesoderm-derived cells,endoderm-derived cells, and others, more specifically to neurons, glia,muscles, cartilage, bone, ciliated epithelium, intestinal epithelium,and the like.

[0221] These results of microscopic examination after HE staining areshown in panels A to H of FIG. 3, and those after immunostaining areshown in panels I to M of FIG. 3.

[0222] Morphological Features:

[0223] 1. There were exhibited high nucleus/cytoplasm ratio, conspicuousnucleoli, and colonization.

[0224] 2. The colonies were more flat, compared to mouse ES cells.

[0225] These morphological features are shown in FIG. 1 and panels A andB of FIG. 2.

[0226] Expression of Cell Surface Markers:

[0227] To confirm the presence or absence of stage-specific embryonicantigens (SSEA), which serve as cell surface markers, immunologicalstaining was carried out by using antibodies against the cell surfacemarkers SSEA-1 (negative control), SSEA-3, and SSEA-4. These antibodieswere obtained from The Developmental Studies Hybridoma Bank of theNational Institute of Child Health and Human Development. Each cellsurface marker for SSEA was evaluated by the following procedures: acell was fixed with 4% para-formaldehyde was reacted with primaryantibody. The cell was then reacted with a labeled polymer (SimplestainPO, manufactured by Nichirei Corporation) prepared by couplingperoxidase and a secondary antibody with an amino acid polymer.Thereafter, Simplestain DAB Solution (manufactured by NichireiCorporation) was added to detect the cell surface markers.

[0228] As a result, SSEA-1 was not detected, whereas SSEA-3 and SSEA-4were detected.

[0229] The results of detection of SSEA-4 by the immunostaining areshown in panel D of FIG. 2.

[0230] Alkaline Phosphatase Activity:

[0231] The alkaline phosphatase activity was determined using HNPP(manufactured by Roche) with Fast-Red TR SaH as the substrate. As aresult, the alkaline phosphatase activity was detected. These results ofdetection are shown in panel C of FIG. 2.

[0232] Industrial Applicability

[0233] The monkey-derived embryonic stem cell of the present inventionis useful for embryological studies, studies of diseases, clinicalapplications, experimental models, and the like on primates, especiallyon humans and monkeys. In addition, since the monkey-derived embryonicstem cell can be obtained in a high yield by the method for producingthe monkey-derived embryonic stem cell, the method is expected to beapplied in embryological studies, studies of diseases, clinicalapplications, experimental models, and the like on primates, especiallyon humans and monkeys. Furthermore, the method for screening a reagentfor specific differentiation to cell or tissue can be utilized in thescreening of a reagent for specific differentiation to cell or tissuewhich is useful for obtaining the desired differentiated cell or thedesired differentiated tissue.

1. A monkey-derived embryonic stem cell obtainable by carrying out aprocess comprising: (a) carrying out fertilization by in vitrofertilization or intracytoplasmic sperm injection using a monkey ovumand a monkey sperm, thereby giving a fertilized ovum; (b) allowing thefertilized ovum obtained in the step (a) to differentiate into ablastocyst by in vitro culture; and (c) establishing an embryonic stemcell line using the blastocyst obtained in the step (b).
 2. Theembryonic stem cell according to claim 1, wherein the monkey is Macacafuscata or Macaca fascicularis.
 3. The embryonic stem cell according toclaim 1, wherein the monkey is Macaca fascicularis.
 4. The embryonicstem cell according to any one of claims 1 to 3, wherein a culturemedium selected from the group consisting of TALP solution, TALP-HEPESsolution and BWW solution is used in the step (a).
 5. The embryonic stemcell according to any one of claims 1 to 4, wherein the in vitro culturein the step (b) is micro-hanging drop culture.
 6. The embryonic stemcell according to any one of claims 1 to 5, wherein the in vitro culturein the step (b) is performed using CMRL-1066.
 7. The embryonic stem cellaccording to any one of claims 1 to 6, wherein the temperature for theculture in the step (b) is at 38° C.
 8. The embryonic stem cellaccording to any one of claims 1 to 7, wherein the conditions for theculture in the step (b) are under conditions at 5% CO₂, 5% O₂ and 90%N₂.
 9. A method for producing a monkey-derived embryonic stem cell,comprising: (a) carrying out fertilization by in vitro fertilization orintracytoplasmic sperm injection using a monkey ovum and a monkey sperm,thereby giving a fertilized ovum; (b) allowing the fertilized ovumobtained in the step (a) to differentiate into a blastocyst by in vitroculture; and (c) establishing an embryonic stem cell line using theblastocyst obtained in the step (b).
 10. The method according to claim9, wherein the monkey is Macaca fuscata or Macaca fascicularis.
 11. Themethod according to claim 9, wherein the monkey is Macaca fascicularis.12. The method according to any one of claims 9 to 11, wherein a culturemedium selected from the group consisting of TALP solution, TALP-HEPESsolution and BWW solution is used in the step (a).
 13. The methodaccording to any one of claims 9 to 12, wherein the in vitro culture inthe step (b) is a micro-hanging drop culture.
 14. The method accordingto any one of claims 9 to 13, wherein the temperature for the culture inthe step (b) is at 38° C.
 15. The method according to any one of claims9 to 14, wherein the conditions for the culture in the step (b) areunder conditions at 5% CO₂, 5% O₂ and 90% N₂.
 16. The method accordingto any one of claims 9 to 15, wherein the in vitro culture in the step(b) is performed using CMRL-1066.
 17. An established cynomolgusmonkey-derived cell having the following properties: (i) being capableof indefinitely proliferating while maintaining an undifferentiatedstate; (ii) having the same chromosome number as that of original Macacafascicularis; (iii) exhibiting the pluripotency when transplanted tohypodermis, the renal capsule or the testis of 8-12 week-old SCID mouseor nude mouse; (iv) being negative for SSEA-1 and positive for SSEA-3and SSEA-4; and (v) exhibiting an alkaline phosphatase activity.
 18. Theestablished cynomologus monkey-derived cell according to claim 17,wherein the cell exhibits the ability to differentiate into at least oneselected from the group consisting of ectoderm, mesoderm and endoderm,when transplanted to hypodermis, the renal capsule or the testis of 8-12week-old SCID mouse or nude mouse.
 19. The established cynomologusmonkey-derived cell according to claim 17 or 18, wherein the cellexhibits the ability to differentiate into at least one selected fromthe group consisting of neuron, glia, muscle, cartilage, bone, ciliatedepithelium and intestinal epithelium, when transplanted to hypodermis,the renal capsule or the testis of a 8-12 week-old SCID mouse or nudemouse.
 20. A method for screening a reagent for specific differentiationinto tissue or cell, characterized by maintaining a cell selected fromthe group consisting of the monkey-derived embryonic stem cell accordingto any one of claims 1 to 8 and the cynomologus monkey-derived cellaccording to any one of claims 17 to 19 in the presence of a testsubstance.
 21. A differentiated cell or differentiated tissue resultingfrom differentiation from a cell selected from the group consisting ofthe embryonic stem cell according to any one of claims 1 to 8 and thecynomologus monkey-derived cell according to any one of claims 17 to 19.